ITI’s Michael Connolly, Manager of Process Development, presents a poster titled, “Functional evaluation of isoforms present in pDNA vaccines” at Aldevron’s corporate offices located in Fargo, North Dakota
A detailed abstract on the poster can be read below…
Title: Functional evaluation of isoforms present in pDNA vaccines
Michael A. Connolly, Jeneice Hamilton and Timothy A. Coleman
Nucleic acid vaccines (DNA or RNA) are experiencing a significant resurgence in the treatment of infectious diseases, allergy and oncology. Plasmid DNA (pDNA) provides the elements required for propagation in a bacterial host (e.g. replication origin, selection marker) but more importantly, they contain all the elements required for directing target-gene expression in human patients. Our technology provides an additional element, the gene encoding Lysosomal Associated Membrane Protein (LAMP) to help traffic the target gene into the MHC-II pathway thus eliciting a strong immune response.
As with other parenteral drugs, pDNAs made for use in clinical studies must meet minimal requirements for safety, identity, purity and potency. One such attribute is Forms Analysis, which relates to the proportion of supercoiled (SC), Open Circular (OC), Linear (L) or other forms like concatamers that are routinely found in the pDNA product. While all these forms are identical in nucleic acid composition, it is generally accepted that preparations containing a higher proportion of SC pDNA will be taken up more readily by cells and ultimately be more efficacious.
In the present study, we prepared different isoforms of pDNA encoding either a GFP reporter or a LAMP fusion gene, confirmed the forms by Agarose Gel Electrophoresis (AGE) and HPLC and analyzed their ability to direct gene expression in vitro (transfection or electroporation) and in vivo (injection into mice). Our results demonstrate that the OC form of pDNA is competent at directing gene expression in vitro. Unexpectedly, a linear form with an intact LAMP fusion gene was also able to direct gene expression. Confirmation of these results in vivo, could be used to substantiate a reduced specification for SC pDNA which would directly impact product yield and manufacturing COGs.